1. Field of the Invention
The present invention relates to a method for detecting bovine diarrhoea virus infection, a nucleotide sequence encoding a protein associated with this virus infection and recombinant proteins and antigens relating thereto.
2. Description of the Prior Art
Bovine diarrhoea virus (BVD) is an infectious single-stranded RNA-containing enveloped virus which is related to the conventional hog cholera virus and to the Border disease virus, the three viruses forming the Pestivirus genus which belongs to the Togaviridae family. The BVD virus is universally distributed in bovine populations and manifests itself by a wide range of clinical symptoms associated with congenital, respiratory or enteric diseases (bovine viral diarrhoea, mucosal disease).
Isolates of BVD viruses may be classified into two distinct categories or biotypes according to their effects during cell culture: cytopathogenic and noncytopathogenic.
Acute infection of seronegative animals is normally benign or subclinical. On the other hand, intrauterine infection of the foetus, during approximately the first four months after the start of pregnancy, by a noncytopathogenic strain can not only produce abortions, still births or the birth of weak calves, but also the birth of calves having persistent viremia, that is to say permanently excreting the virus. This period of four months corresponds to an absence of immunity in the foetus. When the immune system then becomes competent, it recognises the virus as its own and a situration of immunotolerance is established (absence of antibodies). These animals will not be able to survive a subsequent infection by a cytopathogenic strain of homologous BVD virus.
Maintenance of the noncytopathogenic virus within the bovine population is ensured by its slow dissemination following acute infection of seronegative animals and, in particular, by its continual excretion by animals having persistent viremia. (See J. Brownlie et al., Ann. Rech. Vet. (1987) 18:157-166).
A. Fenton et al. (Journal of Virological Methods, 27 (1990), 253-260) detect the Pestivirus antigens in the blood of viremic sheep infected in a persistent manner by the Border disease virus, by an ELISA carried out so as to detect a specific antigen in the leucocytes of these animals. This technique requires prior purification of the leucocytes, which proves to be long and complex to carry out.
The genome of the Osloss viral strain, of cytopathogenic biotype, has been cloned and completely sequenced by Renard et al. (Patent Application EP-A-0,208,672 of 8 July 1985). The Applicant has found that the open reading frame (ORF) of the BVD Osloss genomic sequence, which is 12408 nucleotides in length, has a coding capacity of 3951 amino acids (aas).
In an abstract distributed during the symposium on ruminant infections by Pestiviruses which was held at Hanover on 8 and 9, June 1990, C. Lecomte et al. indicate the identification of a cDNA translational product of the BVD virus immunoprecipitated by monoclonal antibodies recognising the nonstructural protein p80 of a certain number of Pestivirus strains. The same cDNA is expressed in E. coli and the antigen produced is used in competition ELISA to detect anti-BVD antibodies in the bovine serum.
The preparation and the characterisation of a series of monoclonal antibodies have been described by C. Lecomte et al. (Veterinary Microbiology, 23 (1990), 193-201), as well as the use of a fusion protein produced in E. coli as recombinant antigen enabling anti-BVD serum antibodies to be detected in a competition ELISA with the chosen monoclonal antibodies.
In an abstract distributed at the VIIIth internalional Congress of Virology which was held in Berlin on 26 to 31 August 1990, C. Lecomte et al. proposed the use of two ELISA tests for the detection on the one hand of anti-BVD antibodies and, on the other hand, of viral antigens. The anti-BVD antibodies in the serum would be detected by a competition ELISA using a BVD Osloss recombinant antigen p80 produced in E. coli and monoclonal antibodies specifically directed against the p80 protein of a certain number of Pestivirus strains. The second ELISA would be a sandwich type ELISA using two monoclonal antibodies and which would permit the detection of antigens in persistent viremic animals.
However, the Applicant has found that the p80 protein produced in E. coli is not recognised by all the anti-p80 monoclonal antibodies and especially by some of those exhibiting polyspecificity, that is to say reacting towards several or all Pestivirus strains, which undermines the chances of being able to detect in a single operation any infection by any BVD strain, all the more so as the second ELISA, based on the use of two monoclonal antibodies, could also not be sufficiently polyspecific.